Quantitative proteomics reveals regulatory differences in. Assess cell viability by live dead staining 24 hr postencapsulation using a viabilitycytotoxity kit. Autologous chondrocyte transplantation is known as autologous chondrocyte implantation aci in north america. Human chondrocytes osteoarthritis hcoa culture protocol. The primary osteoblast chondrocyte co culture model developed in this study is based on the combined culture of a high density chondrocyte micromass and a mono. In primary cell culture, the 60k collagen appeared in zones 2a, 2b, 3a, and 3b often increasing in concentration in the latter zones. Immortalization of chondrocytes increases life span and proliferative capacity but does not necessarily stabilize the differentiated phenotype.
M e t h o d s i n m o l e c u l a r m e d i c i n e cartilage. Chondrocyte culture has been essential to understand cartilage degeneration, which is a hallmark of oa. Physically entrapped gelatin in polyethylene glycol. The final agarose concentration of chondrocyte agarose. Osteoarthritis is an inflammatory disease characterized by increased degradation of cartilage tissue in the joint due to overproduction of enzymes degrading the extracellular matrix. Original article primary culture and adenoviral infection of. Culture systems that support chondrocyte phenotype include suspension culture in spinner flasks, in dishes coated with a nonadherent substrates 2426, in pellets or micromasses 27, 28, and in threedimensional matrices such as collagen gels, agarose 12, 30, 31, alginate 32, 33, or collagen sponges 34, 35.
Subculture the cells when the hc culture reaches 80% confluency. The extracellular matrix ecm of articular cartilage has several functions that are unique to joints. Initial proof of principle studies could be initiated with the use of in vitro cell culture methods such as 3dimensional cell culture models i. Isolating articular chondrocytes from cartilage for cell culture articular cartilage is located on bone surfaces within vertebral joints, and ensures a smooth and almostfrictionless surface for the movement of the bones within the joint. Chondrocyte culture and assay liebman 2001 current. Method and device development for 3dimensional chondrocyte. Culture of immortalized chondrocytes and their use as models. Enhancement of chondrogenesis via coculture of bovine. Matrixinduced autologous chondrocyte implantation and. This is in agreement with our own results from silac. Autologous chondrocyte implantation aci is a technique that involves the cultivation. Organisation of the chondrocyte cytoskeleton and its response to changing mechanical conditions in organ culture volume 194 issue 3 l. Change chondrocyte growth medium every other day until the cells reach 60% confluency.
University of manchester chondrocyte protocol susan kimber lab. Development of in vitro joint tissue coculture models for. Chondrocyte cultures from human proximal interphalangeal. Culture and phenotyping of chondrocytes in primary culture. Many types of culture exist and have their own advantages and drawbacks. Enhancement of chondrogenesis via co culture of bovine chondrocytes with demineralized bone matrix in chitosanalginate beads bibek chandra sutradhar1 1 1,gyeongmi hong zigang ge2,3 gonhyung kim 1laboratory of veterinary surgery, college of veterinary medicine chungbuk national university cheongju 361763 south korea. The chondrocyte seeded constructs cultured in a serumsupplemented medium have been found to induce cell division and extracellular matrix synthesis and retention for up to several weeks in a variety of scaffolds. Three dimensional chondrocyte cultures on a scaffold are widely used to retain the chondrogenic phenotype. Cultivate cells in high density culture for at least 3 days. Chondrocyte culture is an efficient target system for evaluation of the pharmacological and toxicological effects of drugs acting on cartilaginous tissue.
The cells of cartilage, called chondrocytes, are isolated in small lacunae within the matrix. The primary osteoblast chondrocyte coculture model developed in this study is based on the combined culture of a high density chondrocyte micromass and a mono. The effect of the bioreactor on plla was similar to what has been shown in numerous previous studies using pga. Isolating articular chondrocytes from cartilage for cell. They produce and maintain the cartilaginous matrix, which consists mainly of collagen and proteoglycans. Pubmed comprises more than 26 million citations for biomedical literature from medline, life science journals, and online books. Human chondrocyte cultures as models of cartilagespecific. Although serumsupplemented culture medium has proven sufficient for in vitro studies, the use of animal. View enhanced pdf access article on wiley online library html view download pdf for offline viewing.
Cartilage, chondrocyte, srebp2, col2a1, aggrecan introduction the articular cartilage is composed of chon. However at later passage the cells lose this ability to recover a cartilage extracellular matrixforming phenotype and this clearly re. Culture for approximately 47 days is required before reasonable amounts of total rna may be extracted. Cellbased therapy using chondrocytes for cartilage repair suffers from chondrocyte dedifferentiation. Hyaluronan production and chondrogenic properties of. Cartilage is enclosed by the perichondrium, a dense fibrous layer. Synthesis, characterization and chondrocyte culture of. The culture and adenoviral infection of adult mouse knee articular chondrocyte described here was supposed to provide a powerful tool for deeper researches concerning knee articular chondrocytes. Novartis institute of biomedical research, summit, new jersey.
Previous studies have proven that co culture technique may provide environment that is necessary for stem cell differentiation and promote chondrocyte proliferation. Expansion of chondrocytes for cartilage tissue engineering. Expansion of chondrocyte cell lines in continuous monolayer culture may result in the loss of phenotype, particularly if high cell density is not maintained. Human chondrocytes hc are derived from normal human articular cartilage, where they produce and maintain the extracellular matrix of cartilage, including type ii collagen. Second, chondrocyte sheets generated using temperatureresponsive culture inserts express fn strongly and have excellent adhesive properties, meaning that the. Mechanical properties and structurefunction relationships of human chondrocyteseeded cartilage constructs after in vitro culture.
The effect of stichopus chloronotus aqueous extract on human. A major problem associated with this approach is that chondrocytes undergo dedifferentiation into fibroblastlike cells during expansion. Human chondrocytes hc culture protocol sigmaaldrich. These data have revealed chondrocyte dedifferentiatthat ion progresses with time in expansion culture. Primary culture of immature murine costal chondrocytes.
Attention should be paid to use of the final product composition in. Pellet culture model, 3dimensional alginate cell culture. You can use chondrocytes cultured for up to 10 days or longer. The second phase of the procedure involves the insertion of the chondrocytes into the defect. Therefore, for tissue engineering strategies it is important to consider how. Surprisingly, presence of synovium in co culture helped significantly reduce chondrocyte nitrite production, suggesting release of antiinflammatory factor by the synovium upon cytokine challenge. However, chondrocytes tend to dedifferentiate to fibroblasts when they are subcultured, which is a major problem. Chondrocyte dedifferentiation in expansion culture. Used widely in research, the cells are a gold standard control for cellular reprogramming and differentiation. Bin wang, rujun li, yan ke, yuankun xu, ke tao, dan xing, jianhao lin. Double the chondrocyte growth medium volume when the culture is 60% confluent or for weekend feedings. Recent examples on the web these mice had a lack of a chondrocyte surface protein called the a2a adenosine receptor that brings the adenosine signal inside the cells. Chondrocytes grown in monolayer culture on a solid surface tend to lose their phenotypic.
The pathogenesis of hand oa hoa is elusive and models to study it have not been described. Mechanical properties and structurefunction relationships of human chondrocyteseeded cartilage constructs after in vitro culture jill m. Autologous chondrocyte transplantation is a promising technique for treatment of cartilage defects. In secondary cultures, the 60k collagen was synthesized by zone 1 cells and to a small extent by the tarsus cells in addition to the cells from the other zones. Introduction the ability to differentiate into multiple lineages is a fundamental characteristic of mesenchymal stem cells. Chondrocyte distribution was observed under the optical microscope at 0 h, 4 h and 24 h, respectively. Characterization of chondrocyte scaffold carriers for cellbased gene therapy in articular cartilage repair wei shui,1,2 liangjun yin,1,2 jeffrey luo,2 ruidong li,1,2 wenwen zhang,1,2 jiye zhang,1,2. Phenotype and genotype analysis of primary articular. Isolation and cell culture of primary human chondrocytes.
Culture the cellladen hydrogels and the empty hydrogels for 36 weeks changing to fresh chondrocyte growth media every 2 days before harvesting for analyses. Chondrocytes from different ages and disease states can be cultured with the described method and analyzed for their phenotype, gene expression and biochemical and mechanical properties of the cartilage tissue generated. A previous report has demonstrated the difficulty to incorporate stable isotope labels into proteins in cartilage explant cultures with a relatively small number of identified labeled proteins as a result. Bonassar1,2 1sibley school of mechanical and aerospace engineering, cornell. The exact manner of chondrocyte culture can vary between different centres. Arthritis clinic and research center, peking university peoples hospital, beijing, p. Chondrocyte definition of chondrocyte by merriamwebster. Cell culture techniques university of maryland, college park. Synthesis, characterization and chondrocyte culture of polyhedral oligomeric silsesquioxane poss containing hybrid hydrogels xiuwei pan, a hao gao, b guodong fu, b yun gao a and weian zhang a. Original article effect of simulated microgravity on human. Organisation of the chondrocyte cytoskeleton and its. Presence of meniscus in co culture did not present as significant of an effect as synovium did.
Remove one cryovial of imef cells from the liquid nitrogen cell storage dewar and place on ice for transfer to the cell culture suite. Mechanical properties and structurefunction relationships of. Likewise, the conditions that are needed to induce redifferentiation change as a function of culture expansion. A flow of culture medium was gently added to remove cells which did not sink in the microwells. Hypoxia combined with spheroid culture improves cartilage. Although the cultures may continue to express chondrocyte phenotype e. The harvested chondrocytes were cultured in dulbeccos modi. Cell culture studies of excess vitamin a inhibits the synthesis of chondroitin sulfate by chondrocytes and causes the inhibition of chondrogenesis in the developing embryo which may result in limb malformations. Firstly the defect is prepared by excising all damaged cartilage, to the depth of the. Remove flask of cells from incubator and check them under the inverted microscope at 10x magnification. The relevance of monolayer culture as an experimental model to study the chondrocyte secretome. The culture medium was changed every three days to aid cell growth. Change to fresh chondrocyte growth medium after 24 hours or overnight to remove all traces of dmso.
Cell sheet, cultured cell, articular cartilage, partial thickness defect, chondrocyte, temperatureresponsive culture dish. Primary human chondrocytes in culture and chondrogenesis in human bone marrow stem cells simon r. Hc human chondrocytes cartilage producing cells cell. Modeling chondrocyte differentiation stemcell technologies. Mechanical properties and structurefunction relationships of human chondrocyte seeded cartilage constructs after in vitro culture jill m. Quantitative proteomics reveals regulatory differences in the. The studies have primarily utilised bone marrow derived. The culture of chondrocytes is one of the most powerful tool for exploring the intracellular and molecular features of chondrocyte differentiation and activation. Fluorescent staining and quantitative realtime pcr of chondrocyte spheroids were carried out. Chondrocyte culture and cell distribution chondrocytes were isolated from the knee joints of pigs yorkshire, 1012 months.
This chapter describes strategies for maintaining or restoring differentiated phenotype in established chondrocyte cell lines involving culture in serumfree defined culture medium, in. Three chondrocyte samples, juvenile 6 months, adult 34 years and osteoarthritic 74 years. Equine eq, bovine bo and ovine ov articular chondrocytes from metacarpophalangeal fetlock f, shoulder s and knee k joints were cultured in tissue culture flasks. Pdf primary culture and phenotyping of murine chondrocytes. Primary culture and adenoviral infection of adult mouse knee articular chondrocytes. Cartilage tissue culture is a new biotechnological field that has been recently emerged to provide an alternative cell culture to the treatment of damaged human tissues such as liver, bladder, chondrocytes, etc. Modeling chondrocyte differentiation 1 modeling chondrocyte differentiation. Cells passages 1 and 4 were encapsulated in alginate gel beads and cultured in spinner flasks in chondrogenic and chondrocyte. Ultrastructure typical chondrocytes are ovoid cells ranging in maximum diameter from about 10 tm in articular cartilage to about 30,tm in other hyaline cartilages stockwell and meachim, 1973. Cells passages 1 and 4 were encapsulated in alginate gel beads and cultured in spinner flasks in chondrogenic and chondrocyte growth media. Although cartilage is avascular, gaseous metabolites and nutrients can diffuse through the aqueous phase of the gellike matrix to reach the cells. Original article primary culture and adenoviral infection.
Similar changes may occur in osteoarthrosis muir, 1977. Hardingham uk centre for tissue engineering and wellcome trust centre for cell matrix research, faculty of life sciences. For example, in monolayer culture chondrocytes do not retain all of their specific functions. Influence of species and anatomical location on chondrocyte. An analysis of the collagen synthesized by chondrocyte. Therefore, researchers have considered loss of type ii, and increases in type i collagen expression as indicators of dedifferentiation table 1. Molecular analysis of chondrocytes cultured in agarose in. Culture of immortalized chondrocytes and their use as. Mechanical properties and structurefunction relationships. In the present study, the effects of an integrated threedimensional and dynamic culture on rabbit articular chondrocytes were investigated.
Characterization of chondrocyte scaffold carriers for cell. Although the word chondroblast is commonly used to describe an immature chondrocyte, the term is imprecise. If cells are rounded continue on with changing the media. Method and device development for 3dimensional chondrocyte pellet culture studies natasha mcmasters 1, marcial garmendiacedillos 1, peter schuck 3, julian lui 2, jeffrey baron 2, tom pohida 1 center for information technology 1, nih national institute of child health and human development 2, national institute of biomedical imaging and.
Bmp4 and fgf2 have been experimentally shown to increase chondrocyte differentiation. Weigh all the collagenase p powder and combine into sterile cell culture media and fbs in the following proportions. Although a technique for transplanting cultured chondrocytes has. Embryonic mouse chondrocytes were isolated from the ventral parts of the rib cages of 17. The aim of this investigation was to study the effect of species, anatomical location and exogenous growth factors on chondrocyte proliferation in vitro. Citations may include links to fulltext content from pubmed central and publisher web sites. Human chondrocytes osteoarthritis hc oa are derived from human articular cartilage of donors with oa. Using a biodegradable gelatin scaffold is one option for the cell delivery system, but molecular and histological studies of the method have not yet been done. Cartilage differs from other connective tissues in that the collagen content is almost entirely type ii. This occurrence may limit the chondrocyte expansion and proliferation in an attempt to treat big lesions through aci procedure.
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